The majority of HIV infections occur via mucosal routes (genital, oral or rectal) world-wide. The overall goal of this proposal is to develop a vaccination approach that induces strong HIV-specific humoral and cellular immunity in genital, intestinal and oral mucosae. We hypothesize that vaccines, which elicit strong anti-HIV immunity at these mucosal sites, will prevent infection and rapidly clear infected cells very early at the site of infection and enhance protection. Oral cavity is rich in lymphoid tissue containing antigen presenting cells, T cells and B cells, and provides an excellent opportunity for mucosal delivery of vaccines. However, this route of immunization has been under utilized to deliver vaccines in part due to the presence of proteases in saliva that can degrade vaccines. This is particularly true for protein-based vaccines. Here, we aim to target the oral mucosa for immunization using a needle free device (Syrijet) as an oral vaccine delivery system to induce strong immune responses at oral and intestinal mucosal sites. The syrijet can deliver vaccines into the oral mucosa thus will 1) enhance the delivery of vaccine to local lymphoid tissue that will facilitate uptake by DC and 2) will maintain integrity of the vaccine by preventing degradation by oral proteases. To test our hypothesis, we will use a heterologous prime/boost regimen consisting of CD40L-adjuvanted DNA, MVA and protein vaccines expressing SHIV immunogens. In our preclinical macaque studies we showed that CD40L- adjuvanted DNA/MVA SIV vaccines delivered via IM route provide enhanced protection against intrarectal neutralization resistant SIV challenges. The addition of systemic protein boost adjuvanted with nanoparticle encapsulated TLR7/8 agonist to the DNA/MVA vaccine robustly boosted HIV envelope-specific IgG in serum and also increased antibody in vaginal and rectal secretions. In this proposal, in Aim 1, we will first optimize the method and location of oral vaccine delivery and compare different adjuvants for protein immunogen. We will compare sublingual and buccal immunizations delivered with and without syrijet for induction of strong SHIV- specific humoral and cellular immunity in the oral and gut mucosal tissue. We will also test other mucosal adjuvants flagellin and dmLT for protein immunizations. In Aim 2, using the best delivery method and adjuvant combination that we identify in Aim 1, we will conduct an intrarectal challenge study with clade C SHIV. This study will also focus on understanding the immune correlates for protection. By completion of these Aims we hope to have identified an efficient mucosal vaccination regimen for enhancing protection against HIV.